Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:6.5.1.2 (DNA ligase)
2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A set of covalently closed circular duplex simian virus 40 DNA preparations of varying superhelical densities was prepared by closure of nicked duplex DNA with polynucleotide ligase in the presence of varying amounts of ethidium. The resulting molecules were tested for complex formation with the lysine-rich histone f1. The results confirmed earlier experiments in demonstrating that f1 histone reacts preferentially with superhelical DNA compared to relaxed circular DNA. Furthermore, the extent of the reaction is demonstrated to depend on the superhelical density. At the relatively low ratios of histone to DNA used in these experiments, the product of the interaction of f1 histone with superhelical DNA does not precipitate. At higher ratios of histone to DNA, an insoluble aggregate is formed.
...
PMID:The effect of superhelicity on the interaction of histone f1 with closed circular duplex DNA. 126 27

Sequence analysis of the SalI g region of the genome of a virulent isolate of ASFV (Malawi Lil 20/1) has revealed an open reading frame with the potential to encode a 48 kilodalton (kD) polypeptide which has significant homology with eukaryotic and prokaryotic DNA ligases. This ASFV encoded gene also contains the putative active site region of DNA ligases including the lysine residue which is necessary for enzyme-adenylate adduct formation, but lacks the C-terminal basic region conserved in other eukaryotic DNA ligases. A novel [32P]-labelled potential DNA ligase-adenylate adduct of M(r) 45 kD was observed upon incubation of ASFV infected cell cytoplasmic extracts with alpha-[32P]-ATP and subsequent analysis of products by SDS/PAGE. These data together suggest that ASFV encodes its own DNA ligase.
...
PMID:An African swine fever virus gene with homology to DNA ligases. 161 52

UvrA is the ATPase subunit of the DNA repair enzyme (A)BC excinuclease. The amino acid sequence of this protein has revealed, in addition to two zinc fingers, three pairs of nucleotide binding motifs each consisting of a Walker A and B sequence. We have conducted site-specific mutagenesis, ATPase kinetic analyses, and nucleotide binding equilibrium measurements to correlate these sequence motifs with activity. Replacement of the invariant Lys by Ala in the putative A sequences indicated that K37 and K646 but not K353 are involved in ATP hydrolysis. In contrast, substitution of the invariant Asp by Asn in the B sequences at positions D238, D513, or D857 had little effect on the in vivo activity of the protein. Nucleotide binding studies revealed a stoichiometry of 0.5 ADP/UvrA monomer while kinetic measurements on wild-type and mutant proteins showed that the active form of UvrA is a dimer with 2 catalytic sites which interact in a positive cooperative manner in the presence of ADP; mutagenesis of K37 but not of K646 attenuated this cooperativity. Loss of ATPase activity was about 75% in the K37A, 86% in the K646A mutant, and 95% in the K37A-K646A double mutant. These amino acid substitutions had only a marginal effect on the specific binding of UvrA to damaged DNA but drastically reduced its ability to deliver UvrB to the damage site. We find that the deficient UvrB loading activity of these mutant UvrA proteins results from their inability to associate with UvrB in the form of (UvrA)2(UvrB)1 complexes. We conclude that UvrA forms a dimer with two ATPase domains involving K37 and K646 and that the work performed by ATP hydrolysis is the delivery of UvrB to the damage site on DNA.
...
PMID:Site-specific mutagenesis of conserved residues within Walker A and B sequences of Escherichia coli UvrA protein. 182 50

We have detected the in situ activities of DNA glycosylase, endonuclease, exonuclease, DNA polymerase, and DNA ligase using a novel polyacrylamide activity gel electrophoresis procedure. DNA metabolizing enzymes were resolved through either native or SDS-polyacrylamide gels containing defined 32P-labeled oligonucleotides annealed to M13 DNA. After electrophoresis, these enzymes catalyzed in situ reactions and their [32P]DNA products were resolved from the gel by a second dimension of electrophoresis through a denaturing DNA sequencing gel. Detection of modified (degraded or elongated) oligonucleotide chains was used to locate various enzyme activities. The catalytic and physical properties of Novikoff hepatoma DNA polymerase beta were found to be similar under both in vitro and in situ conditions. With 3'-terminally matched and mismatched [32P]DNA substrates in the same activity gel, DNA polymerase and/or 3' to 5' exonuclease activities of Escherichia coli DNA polymerase I (large fragment), DNA polymerase III (holoenzyme), and exonuclease III were detected and characterized. In addition, use of matched and mismatched DNA primers permitted the uncoupling of mismatch excision and chain extension steps. Activities first detected in nondenaturing activity gels as either multifunctional or multimeric enzymes were also identified in denaturing activity gels, and assignment of activities to specific polypeptides suggested subunit composition. Furthermore, DNA substrates cast within polyacrylamide gels were successfully modified by the exogenous enzymes polynucleotide kinase and alkaline phosphatase before and after in situ detection of E. coli DNA ligase activity, respectively. Several restriction endonucleases and the tripeptide (Lys-Trp-Lys), which acts as an apurinic/apyrimidinic endonuclease, were able to diffuse into gels and modify DNA. This ability to create intermediate substrates within activity gels could prove extremely useful in delineating the steps of DNA replication and repair pathways.
...
PMID:Characterization of DNA metabolizing enzymes in situ following polyacrylamide gel electrophoresis. 200 53

Several DNA-interactive proteins, including the DNA repair enzyme T4 endonuclease V, have been shown to locate their target recognition sites utilizing an electrostatically mediated facilitated diffusion mechanism. Previous work indicates that a decrease in the affinity of endonuclease V for nontarget DNA results in an increased nontarget dissociation rate. This study was designed to investigate the effect of an increase in the affinity of endonuclease V for nontarget DNA. Using a working structural model of the enzyme as a guide, the electrostatic character of endonuclease V was altered. Substitution of Thr-7 with Lys-7 resulted in an enzyme with wild type in vitro characteristics. Mutations which increased the positive charge along a proposed solvent-exposed alpha-helical face had significant effects. The mutants Ala-30, Val-31----Lys-30, Leu-31 and Asn-37----Lys-37 displayed wild type in vitro apurinic-specific and dimer-specific nicking activities. Although the processive dimer-specific nicking rate of the Lys-37 mutant resembled that of wild type, the rate of the Lys-30, Leu-31 mutant was reduced by 60%. In addition, the salt concentration range over which these mutants processively nick dimer-containing DNA has been greatly expanded. Both mutants are shown to have an increased affinity for nontarget DNA.
...
PMID:Substitution of basic amino acids within endonuclease V enhances nontarget DNA binding. 200 4

Nucleotide sequencing of the vaccinia virus SalI F DNA fragment identified an open reading frame of 552 amino acids encoding a protein of 63.3 kDa. The deduced amino acid sequence shares 30% identity with S. pombe and S. cerevisiae DNA ligases, with homology strongest near the carboxy terminus and around the lysine residue required for ligase-adenylate formation. Prokaryotic DNA ligases are poorly related to the vaccinia sequence. The initiation codon of the ORF forms part of a late transcriptional initiation sequence TAAATG and is preceded by two overlapping early transcriptional termination signals, TTTTTTTAT. Nonetheless, RNA mapping showed that the ligase gene is transcribed early during infection and the 5' end of the mRNA maps to the TAAATG motif. The possible roles of a DNA ligase in vaccinia virus DNA replication and recombination are discussed.
...
PMID:Transcriptional mapping and nucleotide sequence of a vaccinia virus gene encoding a polypeptide with extensive homology to DNA ligases. 255 82

The DNA sequence of the bacteriophage T4 denV gene which encodes the DNA repair enzyme endonuclease V was previously constructed behind the hybrid lambda promoter OLPR in a plasmid vector. The OLPR-denV sequence was subcloned in M13mp18 and used as template to construct site-specific mutations in the denV structural gene in order to investigate structure/function relationships between the primary structure of the protein and its various DNA binding and catalytic activities. The Lys-130 residue of the wild-type endonuclease V has been postulated to be associated with its apurinic endonuclease (AP-endonuclease) activity. The codon for Lys-130 was changed to His-130 or Gly-130, and each denV sequence was subcloned into a pEMBL expression vector. These plasmids were transformed into repair-deficient Escherichia coli (uvrA recA), and the following parameters were examined for cells or cell extracts: expression and accumulation of endonuclease V protein (K-130, H-130, or G-130); survival after UV irradiation; dimer-specific DNA binding; and kinetics of phosphodiester bond scission at pyrimidine dimer sites, dimer-specific N-glycosylase activity, and AP-endonuclease activity. The enzyme's intracellular accumulation was significantly decreased for G-130 and slightly decreased for H-130 despite normal levels of denV-specific mRNA for each mutant. On a molar basis, the endonuclease V gene products generally gave parallel levels of each of the catalytic and binding functions with K-130 greater than H-130 greater than G-130 much greater than control denV-.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Site-directed mutagenesis of the T4 endonuclease V gene: role of lysine-130. 313 2

Proteolytic degradation of the Escherichia coli DNA ligase-adenylate intermediate releases adenosine 5'-monophosphate linked to the epsilon-amino group of lysine by a phosphoamide bond. Measurements of the rate of hydroxylaminolysis of the ligase-adenylate provide further support for a phosphoamide linkage in the native enzyme. Lysine (epsilon-amino)-linked adenosine monophosphoramidate has also been isolated from the T4 phage-induced ligase-adenylate intermediate. These results indicate that an initial step of the DNA ligase reaction consists of the nucleophilic attack of the epsilon-amino group of a lysine residue of the enzyme on the adenylyl phosphorus of DPN or ATP that leads to the formation of enzyme-bound lysine (epsilonamino)-linked adenosine monophosphoramidate.
...
PMID:Structure of the DNA ligase-adenylate intermediate: lysine (epsilon-amino)-linked adenosine monophosphoramidate. 494 32

Using kinetic methods and differential spectrophotometry, the interaction between DNA and RNA ligases T4 and cibacron blue F3GA was studied. It was shown that the dye inhibits the first step of the enzymatic reaction, i. e. the formation of the AMP ligase complex. A 50% inhibition of the AMP-ligase complex by DNA ligase occurs at the dye concentration of 1 X 10(-5) M, that by RNA ligase--at 1 X 10(-4) M. Cibacron blue F3GA also inhibits the formation of end products of the reaction catalyzed by these enzymes. The dye is a noncompetitive inhibitor of RNA ligase with respect to [32P]oligoA20 and a competitive one with respect to ATP. Using differential spectrophotometry, it was found that the interaction occurs predominantly via electrostatic bonds between the SH-groups of the dye and the amino groups of lysine residues. DNA ligase possesses a higher affinity for the dye than RNA ligase.
...
PMID:[Interaction of DNA and RNA ligases of phage T4 with Cibacron Blue F 3GA]. 670 48

Pronuclear injection is currently the most often used method to make transgenic animals, but in some animal species it is temporally restrictive due to difficulty in visualizing pronuclei. However, the injection of construct DNA into the cytoplasm does not result in transgenesis. The production of transgenic mice by a cytoplasmic microinjection technique of polylysine complexed DNA into pronuclear stage zygotes is described. Transgenic mice were produced from cytoplasmic microinjection of mixtures of a 5.3 kb linearized DNA and poly-L-lysine (degree of polymerization = 51). Effects on transgenic frequency of both the lysine to phosphate ratio of polylysine to DNA and DNA concentration were studied. About 12.8% of the pups born from zygotes cytoplasmically microinjected with a polylysine/DNA mixture having a lysine to phosphate ratio (L:P) of 1:1 at a DNA concentration of 50 micrograms ml-1 were transgenic. The transgenic frequency for the pronuclear microinjection positive control of DNA alone was 21.7%. No transgenic pups were born from microinjection of DNA alone into the cytoplasm. Complexes of polylysine/DNA were detected using agarose gel electrophoresis at the conditions which produced transgenic mice. The presence of polylysine with construct DNA altered the in vitro activities of restriction endonuclease and DNA ligase on the construct DNA. The production of transgenic animals using DNA and polylysine in the absence of any other signal protein suggests that a DNA/polylysine complex but not DNA alone can act as a substrate for transgenesis from the cytoplasm.
...
PMID:Transgenesis in mice by cytoplasmic injection of polylysine/DNA mixtures. 758 16


1 2 3 4 5 6 Next >>

---